The selectivity filter of the mitochondrial protein import machinery

Abstract

Background The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported. Results We identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore. Conclusions The general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

“…In our experiments, we found that an exchange of this residue for alanine (R192A) reduced the efficiency of Orf9b binding to TOM70 by about 50%. Remarkably, this effect corresponds to similar values with regard to the contribution of TOM70 in the import of proteins into the inner mitochondrial compartments [24,33,34]. For efficient import of nuclear-encoded proteins into mitochondria, TOM70 is not essential; however, it substantially facilitates the targeting of its substrate proteins.…”

“…Using this system, we found that radiolabeled Orf9b associated with TOM70 (Figure 1C, lane 1). Under the same conditions, radiolabeled mouse dihydrofolate reductase (DHFR), a cytosolic protein of 21.6 kDa, often used as an established model protein [24][25][26], showed no binding to the receptor (Figure 1C, lane 2). Signals obtained after SDS-PAGE and autoradiographic detection were quantified using 2% of the total added lysate as internal control ("Load").…”

“…In the cytosol, these preproteins are bound by chaperone proteins and, thereby, retained in a soluble state [18,19]. We recently found that in the mitochondrial import of carrier proteins, TOM70 functions essentially as a co-chaperone [24]. To test for possible effects of Orf9b on this system, we pre-incubated TOM70 with Orf9b WT and subsequently added reticulocyte lysate containing radiolabeled human ADP/ATP carrier 3 (AAC3), a model protein for chaperone-mediated interaction with TOM70.…”

“…Using BN-PAGE, we analyzed samples of the reticulocyte lysate and found that most of the Orf9b was a component of a high molecular weight complex of 220-240 kDa, corresponding to the size of newly synthesized chaperonebound preproteins that are retained in a soluble state for subsequent import into mitochondria [21,22]. In mitochondrial protein import, TOM70 acts as a co-chaperone by binding chaperone-associated preproteins to facilitate the recognition of their targeting signals by the translocase of the outer membrane (TOM) complex [24]. In human TOM70, the interaction site for the recruitment of the chaperone-associated preproteins is defined by an arginine residue in position 192 within the N-terminal part of TOM70 [31].…”

The Diversity of the Mitochondrial Outer Membrane Protein Import Channels: Emerging Targets for Modulation

“…Tom40 is the central and channel-forming subunit of the TOM complex [ 3 , 21 ]. A wide range of important studies on protein import into mitochondria have shown that Tom40 is not only the entry gate for most mitochondrial precursor proteins delivered from the cytosol to various sub-mitochondrial locations [ 39 , 40 , 41 ] since it also works as a decisive general selectivity filter in the uptake of newly synthesized mitochondrial proteins [ 42 , 43 ]. Moreover, it was also proposed that the TOM complex can act as an insertase mediating lateral release of the membrane imported proteins into the outer membrane [ 44 ].…”